The overall goal of this proposal is to identify genetic and epigenetic alterations occurring at gene enhancer elements in colon cancer, and to gain insights into how these events mediate disease progression. Cancer is due to the progressive accumulation of mutations in DNA, as well as heritable changes in gene expression caused by mechanisms other than mutations in the underlying DNA sequence, so called epigenetic alterations. Gene enhancer elements are short regions of DNA to which transcription factors bind in order to increase the expression of a gene. Enhancers are almost certainly altered at both the genetic and epigenetic level in cancer, although the extent by which this occurs is unknown. Using state of the art ChIP-seq technology, we identified the locations of gene enhancer elements across the entire genome in cells derived from human colon cancer and normal colon. While the locations of many enhancers remain unchanged, thousands of enhancer loci differ between normal colon and colon cancer cells. We call these regions variations at enhancer loci, or VELs. We hypothesize that VELs, which either contain somatic mutations or are purely epigenetically derived, contribute to the formation and progression of colon tumors. Three specific aims are proposed. In Aim 1 we will systematically assess the relationship between VELs and colon cancer progression through characterization of VELs in a cohort of well-characterized primary cell lines that comprehensively capture all of the clinical stages of colon cancer. In Aim 2, we will conduct DNA sequencing of VELs in tumor and matched normal DNA from ten colon cancer patients to identify somatic mutations that may have accrued at gene enhancer elements during carcinogenesis. In Aim 3, we will assess the plasticity and reversibility of the VELs through innovative experiments in which colon cancer cells are reverted to the embryonic state and then re- differentiated into colon epithelium. The successful completion of these Aims will accelerate our understanding of epigenetics and the role of a class of non-coding functional elements in colon cancer, which could ultimately have important implications for therapeutic studies aimed at targeting restoration of aberrantly expressed genes in colon cancer.